The OsNAC41-RoLe1-OsAGAP module promotes root development and drought tolerance in upland rice.

Drought is a major environmental stress limiting crop yields worldwide. Upland rice (Oryza sativa) has evolved complex genetic mechanisms to adjust to drought stress. However, few genetic variants have been identified that mediate drought resistance in upland rice, and little is known about the evolution of this trait during domestication. Here, using a genome-wide association study in rice, we identified ROOT LENGTH 1 (RoLe1) controlling root length and drought resistance. We demonstrate that a G-to-T polymorphism in the RoLe1 promoter increases binding of the transcription factor OsNAC41 to activate its transcription. We also show that RoLe1 interacts with and interferes with the function of OsAGAP, an ARF-GTPase activating protein involved in auxin-dependent root development, to modulate root development. Furthermore, RoLe1 enhanced crop yield by increasing the seed setting rate under moderate drought conditions. Genomic evolution analysis showed that a newly arisen favorable allelic variant, proRoLe1-526T, originated from Region I (Midwest Asia) and was retained in upland rice during domestication. Our findings propose a OsNAC41-RoLe1-OsAGAP module, providing promising genetic targets for molecular breeding of drought-resistant varieties in rice.

QTL detection and candidate gene identification of qCTB1 for cold tolerance in the Yunnan plateau landrace rice.

Cold stress is one of the main abiotic stresses that affects rice growth and production worldwide. Dissection of the genetic basis is important for genetic improvement of cold tolerance in rice. In this study, a new source of cold-tolerant accession from the Yunnan plateau, Lijiangxiaoheigu, was used as the donor parent and crossed with a cold-sensitive cultivar, Deyou17, to develop recombinant inbred lines (RILs) for quantitative trait locus (QTL) analysis for cold tolerance at the early seedling and booting stages in rice. In total, three QTLs for cold tolerance at the early seedling stage on chromosomes 2 and 7, and four QTLs at the booting stage on chromosomes 1, 3, 5, and 7, were identified. Haplotype and linear regression analyses showed that QTL pyramiding based on the additive effect of these favorable loci has good potential for cold tolerance breeding. Effect assessment in the RIL and BC3F3 populations demonstrated that qCTB1 had a stable effect on cold tolerance at the booting stage in the genetic segregation populations. Under different cold stress conditions, qCTB1 was fine-mapped to a 341-kb interval between markers M3 and M4. Through the combination of parental sequence comparison, candidate gene-based association analysis, and tissue and cold-induced expression analyses, eight important candidate genes for qCTB1 were identified. This study will provide genetic resources for molecular breeding and gene cloning to improve cold tolerance in rice. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01488-3.

Cold-adaptive evolution at the reproductive stage in Geng/japonica subspecies reveals the role of OsMAPK3 and OsLEA9.

Cold stress at the reproductive stage severely affects the production and geographic distribution of rice. The Geng/japonica subpopulation gradually developed stronger cold adaptation than the Xian/indica subpopulation during the long-term domestication of cultivated rice. However, the evolutionary path and natural alleles underlying the cold adaptability of intra-Geng subspecies remain largely unknown. Here, we identified MITOGEN-ACTIVATED PROTEIN KINASE 3 (OsMAPK3) and LATE EMBRYOGENESIS ABUNDANT PROTEIN 9 (OsLEA9) as two important regulators for the cold adaptation of Geng subspecies from a combination of transcriptome analysis and genome-wide association study. Transgenic validation showed that OsMAPK3 and OsLEA9 confer cold tolerance at the reproductive stage. Selection and evolution analysis suggested that the Geng version of OsMAPK3 (OsMAPK3Geng ) directly evolved from Chinese Oryza rufipogon III and was largely retained in high-latitude and high-altitude regions with low temperatures during domestication. Later, the functional nucleotide polymorphism (FNP-776) in the Kunmingxiaobaigu and Lijiangxiaoheigu version of the OsLEA9 (OsLEA9KL ) promoter originated from novel variation of intra-Geng was selected and predominantly retained in temperate Geng to improve the adaptation of Geng together with OsMAPK3Geng to colder climatic conditions in high-latitude areas. Breeding potential analysis suggested that pyramiding of OsMAPK3Geng and OsLEA9KL enhanced the cold tolerance of Geng and promotes the expansion of cultivated rice to colder regions. This study not only highlights the evolutionary path taken by the cold-adaptive differentiation of intra-Geng, but also provides new genetic resources for rice molecular breeding in low-temperature areas.

Differentiation, evolution and utilization of natural alleles for cold adaptability at the reproductive stage in rice.

Genetic studies on cold tolerance at the reproductive stage in rice could lead to significant reductions in yield losses. However, knowledge about the genetic basis and adaptive differentiation, as well as the evolution and utilization of the underlying natural alleles, remains limited. Here, 580 rice accessions in two association panels were used to perform genome-wide association study, and 156 loci associated with cold tolerance at the reproductive stage were identified. Os01g0923600 and Os01g0923800 were identified as promising candidate genes in qCTB1t, a major associated locus. Through population genetic analyses, 22 and 29 divergent regions controlling cold adaptive differentiation inter-subspecies (Xian/Indica and Geng/Japonica) and intra-Geng, respectively, were identified. Joint analyses of four cloned cold-tolerance genes showed that they had different origins and utilizations under various climatic conditions. bZIP73 and OsAPX1 differentiating inter-subspecies evolved directly from wild rice, whereas the novel mutations CTB4a and Ctb1 arose in Geng during adaptation to colder climates. The cold-tolerant Geng accessions have undergone stronger selection under colder climate conditions than other accessions during the domestication and breeding processes. Additive effects of dominant allelic variants of four identified genes have been important in adaptation to cold in modern rice varieties. Therefore, this study provides valuable information for further gene discovery and pyramiding breeding to improve cold tolerance at the reproductive stage in rice.

Interaction between serine carboxypeptidase-like protein TtGS5 and Annexin D1 in developing seeds of Triticum timopheevi.

GS5 encoding a serine carboxypeptidase-like protein positively regulates grain size and weight through the regulation of grain width and filling and is helpful in improving cereal yields. Grain width variation determined by GS5 is associated with cell number and size, but the actual underlying mechanism is still unclear. Two orthologs of GS5, TtGS5-3A-G and TtGS5-3G-G, were cloned from the Triticum timopheevi accession no. CWI17006. To identify the proteins that interacted with TtGS5-3A-G and TtGS5-3G-G in premature grains, we performed pull-down assays followed by liquid chromatography-mass spectrometry/mass spectrometry analysis. The analyses revealed 18 proteins were present in both the TtGS5-3A-G and TtGS5-3G-G interactomes. Among five candidates selected, only Annexin D1 interacted with both TtGS5-3A-G and TtGS5-3G-G in yeast. Annexin D1, TtGS5-3A-G, and TtGS5-3G-G were located on the cytoplasmic membranes of Arabidopsis protoplasts and onion epidermal cells, and interactions between Annexin D1 and TtGS5-3A-G, as well as TtGS5-3G-G, were shown by bimolecular fluorescence complementation assays. Annexin D1 was expressed widely in different tissues, and it co-expressed with TtGS5-3A-G/TtGS5-3G-G at the grain enlargement phase. These results indicated that Annexin D1 interacted with TtGS5-3A-G and TtGS5-3G-G in premature grains. Together with the structural similarities of Annexin D1 to known fiber elongation factors, we proposed that TtGS5 might regulate the cell size by interacting with Annexin D1. The results provide significant new information for understanding the roles that GS5 plays in regulating grain size, which may be useful in improving crop yields.

Analyzing the action of evolutionarily conserved modules on HMW-GS 1Ax1 promoter activity.

KEY MESSAGE: The specific and high-level expression of 1Ax1 is determined by different promoter regions. HMW-GS synthesis occurs in aleurone layer cells. Heterologous proteins can be stored in protein bodies. High-molecular-weight glutenin subunit (HMW-GS) is highly expressed in the endosperm of wheat and relative species, where their expression level and allelic variation affect the bread-making quality and nutrient quality of flour. However, the mechanism regulating HMW-GS expression remains elusive. In this study, we analyzed the distribution of cis-acting elements in the 2659-bp promoter region of the HMW-GS gene 1Ax1, which can be divided into five element-enriched regions. Fragments derived from progressive 5' deletions were used to drive GUS gene expression in transgenic wheat, which was confirmed in aleurone layer cells, inner starchy endosperm cells, starchy endosperm transfer cells, and aleurone transfer cells by histochemical staining. The promoter region ranging from - 297 to - 1 was responsible for tissue-specific expression, while fragments from - 1724 to - 618 and from - 618 to - 297 were responsible for high-level expression. Under the control of the 1Ax1 promoter, heterologous protein could be stored in the form of protein bodies in inner starchy endosperm cells, even without a special location signal. Our findings not only deepen our understanding of glutenin expression regulation, trafficking, and accumulation but also provide a strategy for the utilization of wheat endosperm as a bioreactor for the production of nutrients and metabolic products.

Genome-wide identification of internal reference genes for normalization of gene expression values during endosperm development in wheat.

Internal reference genes that are stably expressed are essential for normalization in comparative expression analyses. However, gene expression varies significantly among species, organisms, tissues, developmental stages, stresses, and treatments. Therefore, identification of stably expressed reference genes in developmental endosperm of bread wheat is important for expression analysis of endosperm genes. As the first study to systematically screen for reference genes across different developmental stages of wheat endosperm, nine genes were selected from among 76 relatively stable genes based on high-throughput RNA sequencing data. The expression stability of these candidate genes and five traditional reference genes was assessed by real-time quantitative PCR combined with three independent algorithms: geNorm, NormFinder, and BestKeeper. The results showed that ATG8d was the most stable gene during wheat endosperm development, followed by Ta54227, while the housekeeping gene GAPDH, commonly used as an internal reference, was the least stable. ATG8d and Ta54227 together formed the optimal combination of reference genes. Comparative expression analysis of glutenin genes indicated that credible quantification could be achieved by normalization against ATG8d in developmental endosperm. The stably expressed gene characterized here can act as a proper internal reference for expression analysis of wheat endosperm genes, especially nutrient- and nutrient synthesis-related genes.

Effects of high-molecular-weight glutenin subunit combination in common wheat on the quality of crumb structure.

BACKGROUND: High-molecular-weight glutenin subunits (HMW-GSs) have important effects on bread-making quality. Allelic variations of HMW-GS in bread wheat varieties contribute in different ways to dough properties and bread volume. However, no systematic analysis has been done on the effects of allelic variation on bread-crumb structure, an important parameter when evaluating bread-making quality. In this study, seven Glu-1 deletion lines and one intact line harboring different encoding loci and derived from a cross between two spring wheat cultivars were used to investigate the contribution of a single Glu-1 locus, or combination of Glu-1 loci, to the crumb structure. RESULTS: Deletion of HMW-GS locus combinations resulted in a decline in slice size, brightness, and fineness of the bread crumb. A desirable crumb structure correlated well with preferred subunit combinations: high levels of GMPs, superior dough properties, and loaf volume. The effects of the HMW-GS combinations were ranked as Dx5 + Dy10 > Bx17 + By18 > Ax1 + Null. The Ax1 + Null allele affected the crumb structure by interacting with the Bx17 + By18 or Dx5 + Dy10. CONCLUSION: High-molecular-weight glutenin subunits had significant effects on the loaf volume and crumb structure; varying effects from different subunit combinations were observed. © 2018 Society of Chemical Industry.